Processing of hydrocarbons



United States Patent C i 2,697,061 PROCESSING on HYDROCARBONS Rhett G. Harris', Charleston; S. C., and Raymond strawinski, Long Beach, Calif., assignors to Texaco Di'lopmen't' Corporation, New York, N. Y., a corporation of Delaware No- Drawing. Application August 17 1950", Serial No. 180,092

8-laiins; (CL 1 95'1) This:invention -relates to the pronactionof biosyh'thetics from hydrocarbons and more particularly to the production of fat-like'saponifi'abha mate'r ials,-- organic acids" and antibiotic substances from tiyd'roear bo'ns'by the} action of microorganisms such as molds-g yeasts amt the like;

It has been -fk nownfor sometime that such ganisms' havetheability 'to attackor metabolize so' ems; Since carbohydrates are relatively expensive raw materials and 1n most cases, of

terial suchas carbohydrates;

greater value than the products produced therefrom; other rawmaterials have been sought. 7 1 n amount of work h'as' been done' withhydrocarbonsbut to date it' has not -been generallysuccessful aiid=ha'sjn0tfb'een fectively utilized in' biosy'nthesisf including the production of 'fat-like saponifiable materials, organic acids and a'ntibiotic substances:

mother I object of" tliis= -inv'ention is the "provision of a' series-"t" novelproduct's"- from" hydrocarbons including fat-like saponifiable materials, organic acids-and antibiotic substances.

Still another object of the invention is the provision A'- considerable 2,697,061" Patented Dec. 14, 1954 2 range of 178 6. 261 C; was fractionated into the 01 lowingqmz Cut Control or Charge Cl the aboye cut s of jzerosinqbuti l wasiouiidp-to give the best yieldandproduct, asexpressedingrahis bimold mycelia. Cut Dggave a yieldaof. over. twice as;.nf1 uch as Cut- A, about 50% more than Cut B and about 33% more of -"noveh' method's for recovering, such synthesized 'prod- Gther objects and advantages of the invention will at) pear'from the followingdescr'iption taken in connection with the attachedclaims: a

ditio'ns'; microorganisms f such" as" molds; yeasts and: the like can'be caused to grow quite rapidly' upon relatively inexpensive substrates such as hydrocalrbons with the production. ofTrelatively large yields of; desirable products including fat-1ike saponifiable materials; antibiotic substances .and organic acids: Such conditions include such factorszas theselection'andmaintenance of=a proper-pH range and temperature'range; and the sele'ction of proper nutrient salts; lnz'some' cases, I the selection of predetermined concentrations of: both salts and substrates is' 'desir'ablet- Broadly stated, the practice'o'f the invention'involves' themseof a nutrient salt solution such as asolution' of the'mineral .salts type with a pH in a selected range and the-use'ofa hydrocarbon, the inoculation'of the mixture'withza selected microorganism or microorganisms; the maintenance of the mixture within pre'det'ermined' from 'oiltanksi Of the abov'eye'ven better resultshaye been obtained with "certain sele'cte'd" cuts'l For 'infs'tance;

a kero'siriewate'r white oil, sold'b yflhe TexasCompan'y under the trademanie.Crystalite and halving la'=B. P.

gasoline, gas oil-, 'lubricatingoils and"bottonrsediments Commercial st ye oil, having a B P. range of,.1'617 C. 27:77. 0 s' mi1ar y..f a .ou. ed by. A. ,S. T; M. distillation methods into the following four cuts:

1 B.,P., (Refraction S aeifi o "0. 1 Index G r a'v v' F. ,m 221 1 1. 4541 M279 G 221L235 ,1 114533 v 0 .8 3,51 H 235452" 124631 0,8115 I. 1252+ 114681! 0'. 8506: Control or Charge.. 3 167-277; a 1. 4590 I 0.83M

'e ei, .cyslqp ntkmai".b ylb nze p 2 3" iinethylp'entane; z pente a a Ty yeasts include Mo'niliwmilr'mamicd; M oniliasitofihil'e "and Saccharomyces.

Thema ority of the above were, secured and" area i able' 'from theAm'e"rican Type Culture Collection. Qnfe;v such mold that is .jsuitablefor use"wasunidentified'except' for the"n' 1n'1ber'9577;" a .onefot 'the best microorganisms for useiin thepro 'si of this invention has been found to be Aspergillus" fume. gangs strain TDC No.,946) which"has"been identified as follo'wsr v H 4 H 1 Source of is0lat'ion.--'A mixe'd'sarnp'le composed of soils taken from areas in which oils were undergoing decomposition and water taken from sum'ps, tank bottoms, and waters which hadbeen in contact with refinery wastes; de omposing" asphalt wax and" used motor" oil were" 111'- cludedinthesample." y,

Date of isolation; October"1, 1947.'

Location of" isolatioj Signal,@ Hill Texas Company, Lq gBeaeh; California p I edia "e s ,fo'ri. Mo awk-A y ro n o -fifiri., 1 salts'medium con'1posedof5% Arabian crudeo'il (topped) and the following medium at pH 4.5.

TH Ja OV ea enerally designated Turfi tt s Y east'and M I i m- H a 1 Conditions of 1'is0lati0n.--Aerobic, at 40 C? Laboratory The Microscopic examination and a comparison of the essential morphological features with those of molds described in the literature indicate that the mold belongs to the fumigatus group of the Aspergilli, the type species of which is Aspergillus fumigatus. A complete description thereof can be found in A Manual of the Aspergilli by Charles Thom, The William and Wilkins Company, Chapter X, pp. 148151.

The character of the nutrient media or mineral salts solution will vary to some extent in accordance with the type of microorganism used and the type of hydrocarbon. Generally, it should contain N, P, K, S, Fe, Mg, the

general composition being well known in the art. A

typical medium is as follows:

Compound: (z

(NI-LQzSOi "grams/liter" 2.0 K2HPO4 d 2.0 NaHzPOi. do 1.0 MgSO; do 0.25 MnClz do 0.01 CaClz do 0.01 FeSO4 do 0.01 Kl do 0.00001 CuSOi do 0.00001 Distilled water milliliters 1000 In some cases, the medium may be fortified or enriched with accessory growth factors such as water extract of yeast as Saccharomyces cerevisiae.

It is to be understood that the character of the medium may also be varied dependent upon the type of product that it is desired to produce in the most abundance.

As regards the mechanical or contact aspect, the process may be practiced in a number of different ways. In its simplest form, the growth may be accomplished in a static phase on the surface of the liquid mixture.

In the alternative, it may be practiced as a deep culture process by mechanical agitation such as stirring or The pH with molds may vary from 1 to 9. With yeasts, the 'pH may vary from 4 to 9. Its selection is governed by the particular microorganisms, the particular hydrocarbon and the end products desired.

The same is true of temperature, the range being from 20 to 4-5 C. The actual time of incubation, usually from 7 to 12 days, is determined by the rate of growth of the microorganisms and the rate of production of the desired lay-products. This is governed by the ability of the enzymes produced by the microorganisms to effect production of the desired products.

As typical examples of the methods of the present invention, attention is called to the following.

Example I A. fumigatus (Strain TDC No. 946), previously identified, kerosine cuts of the types previously identified as cuts A, B, C, and D (about two parts by volume) and a nutrient medium (about 30 parts by volume) were combined under incubating conditions at a temperature of 28 to 30 C. for about one week. The nutrient medium in this case was of the following approximate composition:

Compound:

Nal-lzPOrlHzO grams/ liter- 1.0 K2HPO4 do 2.0 MnClzAI-lzO -do 0.01 CaClzZHzO do 0.01 KI do 0.00001 (NH4)2SO4 do 2.0 MgS04.7I-I2O do 0.25 FeSO4.7H2O d0 0.01 CuSO4.5H2O do 0.00001 Sea water milliliters 10.0 Distilled water do 990.0

The pH of the above medium was adjusted to about 5.5 because of it being a good pH value for mold growth and a good pH value for inhibiting bacterial contamination.

After incubation for about one week, the growth was harvested by straining and squeezing to remove excess hydrocarbon and medium. The solid material was then dried at C. to C. for about twenty-four hours, extracted with chloroform, and the fat-like saponifiable material taken from the chloroform in a yield of about 11%.

Referring more specifically to the method of extraction, the mycelia were harvested as by straining and squeezing, dried and a crude extract prepared by chloroform extraction. After removal of the solvent, the residue was treated with approximately two-thirds its weight of potassium hydroxide dissolved in alcohol and refluxed to effect saponification. Following saponification, about twothirds of the solvent alcohol was removed by heating. The residual soap solution was then extracted with petroleum ether to remove the non-saponifiable fraction. Following this step, the aqueous liquor was made strongly acid with a mineral acid and the free organic acids extracted with diethyl ether.

The saponifiable material thus recovered does not consist exclusively of fatty acids of the usual aliphatic character. Some products were found which reacted chemically very nearly the same as the fatty acids but they were not the usual aliphatic fatty acids such as the straightchain acids containing an even number of carbon atoms such as C10 acid (capric), C12 acid (lauric or dodecenoic), etc., such acids being usually obtained from the naturally occurring fats.

While the time of incubation can be continued for a longer period of time, it has been found that there is only a slight increase in yield on continuing incubation even to the extent of an additional week and incubation beyond that time tends to result in a reduction of yield.

The mixed fatty acids obtained from the above procedure are in the nature of pale yellow solids at room temperature with the characteristic appearance of a fat. A slightly, pleasant, fat-like odor is present and the taste is similar to that of candle tallow or similar stearins. The product melts at about 29.5 C. and has a setting point of C. The mean molecular weight is 294, and the iodine number, 83.5. Of the common fats or fatty oils,

the product most closely resembled the mixed fatty acids from olive oil.

Example 11 As an example of an application of the method to the deep-culture technique wherein the reactants are in contact throughout the body of the mixture rather than at an interface, a container was provided with suitable stirring means of a type such that the stirring speed could be controlled. A supply of sterile air was connected so that it could be distributed through a hollow stirring rod and through porous diffusion bulbs attached to the stirring arms. Thirty parts by volume of the nutrient medium above-described in connection with Example I at a pH of about 5.0 was mixed with 2 parts per volume of hydrocarbon such as kerosine and an inoculum of A. fumigatus (Strain TDC No. 946) was added. The inoculum was formed as a thick suspension of the organism in nutrient medium constituting about 10% by volume of the total charge. Either air or relatively pure oxygen can be used as the aerating medium. The mixture was stirred constantly for about one week at 28 C. to 30 C. The liquid and the mycelia were separated by a centrifuge and the myceliurn treated as described in Example l. Part of the water phase was concentrated and the acids contained therein extracted by ether extraction. Another part of the water phase with the mold removed and not concentrated was sterilized by a bacterial filter (cold sterilization) and tested as later described.

Example III of the Petri plate with sterile forceps.

apa t-met 1 broIwnnliquid with a disagreeable odor. on distillation and continued heating, there is "evidence-of decomposi tio'n wtihout additional distillate, the residue bein'g dark, very viscousliquid, insoluble in waterfgivin :an acid reaction, in. bicarbonat After treatment v methanol, the ester was distilled under vacuum and "the distillate was saponified. After acid'ificationflthe free acids were removed by extraction. The resultant material dissolved in both alkali and bicarbonate and exhibited a neutral equivalent of 209.

Further analysis according to the method of Baker and Barkenbus Lind. Eng. Chem., Anal. Ed. 9, 135 (1937)] which is a fusion with a potassium carbonatemagnesium mixture indicated the absence of sulfur, nitrogen and halogens.

When the ether extract is saponified, the soap extracted to remove possible non-saponifiables, and after acidification, the acid material recovered by ether extraction, the acid recovery is about 42% of the original sample.

Such acid material is a viscous liquid at room temperature, does not crystallize at C. and has a neutral equivalent of 153. When just neutralized with NaOH to form the sodium soaps and treated with a solution of copper sulfate, a voluminous green-blue precipitate is produced. This copper salt, after isolation and drying .w1ll ignite but is insoluble in diethyl ether, petroleum ether, pentane or gasoline.

The iodine number of the acid material is 33. When dissolved to make a solution in acetone, it will not crystallize at temperatures as low as -65 C.

In separating the antibiotic substance, both the microorganisms and the residual hydrocarbon were removed from the liquid by the Sharples super-centrifuge. By a proper choice of califier rings, which choice will vary with the system being handled, it is possible to make a three-way separation into oil, aqueous phase, and solids, the latter containing organisms and precipitated salts. Removal of all finely divided materials and sterilization of the liquor can be accomplished by filtering under suction with a Berkereld or oelas ,type filter.

In testing for antibiotic actlon, test organisms were inoculated into nutrient broth and incubated at 30 C. for forty-eight hours. A long incubation period was used because of the slow growing characteristics of some of the organisms that were used and the desire to bring such organisms to their most efiective stage. Organisms used included Pseadomonas aeruginosa, No. 10147 from the American Type Culture Collection, Escherichia coli, No. 9637 from the American Type Culture, Mycobacterium phlei, No. 10142 from the American Type Culture Collection, Mycobacterium smegamatis, No. 10143 from the American Type Culture Collection and Bacillus cereus (mycoides), No. 9634 from the American Type Culture Collection.

An inoculum of 0.1 ml. of the broth culture was added to ml. of sterile, melted nutrient agar at 40 C. to 45 C. in separate test tubes, mixed, and poured into separate Petri plates. After the agar had become solid, circular pieces of sterile filter paper, about /r." in diameter, were dipped into the liquor to be tested, allowed to drain free of excess liquid, and placed on the surface The plate with the adsorbed test papers was then inverted and incubated at C. for forty-eight hours. In every case, evidence of antagonistic action was shown by inhibition of the growth of the test organism, i. e., the formation of a clear zone surrounding the test paper adjacent to the test paper.

As a result of the above test, it was found that the sterile filtrates were antagonistic to Bacillus sabtilis, a Gram-positive spore-forming bacillus, which is a common inhabitant of soil, is non-pathogenic and produces an antibiotic subtilin; Bacillium cereus, a Gram-positive,

soluble in dilute alkali and partially-so ublp under 'csterif-ying conditio s with.

yield ran only about 7.8% of the dry mycelia.

smegmatls or the smegma bacillus, "anacidefast, nonspore formi'rig bacillus "belonging .to thesame genus as a tubercle bacillus (M-ycobac'triury tuberculosis) and characterized by a thick waxelike coating on the surface of its cell which makes it highly impervious to water or water-soluble substances; and Rseudomonas aerugiizosa, a Gram-negative, non-spore-forming bacillus which under some 'co'ndi'tions is of pathogenic significance and produces a characteristie-blue pus "in"fcontaniinated wounds. The positivetest demonstrated with respect to the last-named microorganism is particularly significant since there are 'few -anta'gjonist-icsubstances or antibiotics capable of antagonistic-action against such a microorganism.

The antagonistic water-soluble substances from the growth of A. famigatas and Monilia murngartica in kerosine were found'to be par-ticularlyeffective against the above-describgd organisms.

In yields, the dried mycelia has been found to produce a consistent return of more than 21% of the crudeextracticontaining the desiredlipid materials in contrast to growth on dextrose under the same conditions wgenfthe n urther refining with the removal of non-saponifiables, fatlike materials amounting to about 10% of the dry mycelia are obtained.

The use of surface-tension active agents has been found to increase the yields to some extent. In one case wherein A. fumigatus (Strain TDC No. 946) was used with kerosine, Span 80 (sorbitan monooleate obtainable from the Atlas Powder Co.) was added. A dilution of the surface-tension depressant was prepared in a degree of 1-80,000 and kerosine added thereto. The dilution was used in a proportion of parts by volume with about 4.5 parts of kerosine. The resultant mixture was inoculated with 10 parts by volume of a thick suspension of the mold and incubated at 30 C. for about twelve days. The yield was increased about 30%. Other surface-active agents such as Roccal (Alkyl dimethyl benzyl ammonium chloride obtainable from Winthrop- Stearns, Inc.), sodium oleate, Tween 20 (sorbitan monolaurate polyoxyalkene derivative obtainable from Atlas Powder Co.) may also be used. The above represent typical surface-tension active agents of the cationic, anionic and non-ionic types.

Increases in yield have also been found where the inoculum is washed. Where 10% of an unwashed inoculum such as A, fumigatus (Strain TDC No. 946) was added to kerosine and a mineral salts medium, the yield was not as high as in a case wherein the mold was previously washed with mineral salts medium. ,Washing can be accomplished by thoroughly soaking the mold in freshly prepared mineral salt medium, draining off the excess liquid, and, by manual pressing, freeing the remaining liquid from the solid mold. Preferably, washing is repeated two or three times, the mold being finely suspended in the minerals salts solution. When washed inoculum was used, as compared with unwashed inoculum, an increase in yield of about 47% of the mold mycelia was obtained.

From the above it is believed apparent that the present invention provides a novel process for the synthesis of relatively valuable fat-like saponifiable materials, organic acids and antibiotics from relatively cheap raw materials, i. e. hydrocarbons.

Obviously many modifications and variations of the invention, as hereinbefore set forth, may be made without departing from the spirit and scope thereof and, therefore, only such limitations should be imposed as are indicated in the appended claims.

We claim:

1. A method of synthesizing fat-like, saponifiable materials and organic acids from hydrocarbons comprising the steps of dispersing hydrocarbon in the substantial absence of other organic substrate in an inorganic nutrient medium for microorganisms, inoculating the resulting mixture with a microorganism selected from the group consisting of Penicillium molds, Aspergillus molds, Monilia yeasts and Saccharomyces yeasts, subjecting the inoculated mixture to incubating conditions for a sustained period of time, collecting the resulting solid material and separating therefrom synthesized fat-like saponifiable materials and organic acids.

is inoculated with Penicillium notatum.

3. A process according to claim 1 in which the mixture is inoculated with Aspergillus fumigatus.

4. A process according to claim 1 in which the mixture is inoculated with Mom'lia murmanica.

5. A process according to claim 1 in which the mixture is inoculated with Aspergillus flavus.

6. A process according to claim 1 in which the mixture is inoculated with Pencillium glaucum.

7. A process according to claim 1 in which the inoculated mixture is incubated at a pH of about 1 to 9 and at a temperature in the range of 20 to 45 C.

8. A a procces according to claim 1 in which the inoculated mixture is incubated in the presence of a surface tension depressant.

References Cited in the file of this patent UNITED STATES PATENTS Name Da Number v Name Date 1,835,998 Giron Dec. 8, 1931 2,171,198 Urbain Aug. 29, 1939 2,171,200 Urbain Aug. 29, 1939 FOREIGN PATENTS Number Country Date 270,349 Great Britain of 1928 OTHER REFERENCES Bushnell et al., J. Bact., 41, 653-73 (1941)cited in Chemical Abstracts vol. 35, page 4796.

Zobell et al., Bull. Am. Assoc. Petroleum Geol. 27, 117593 (1943)-cited in Chemical Abstracts V01. 37, page 6693.

Florey et al., Antibiotics, Oxford Medical Publications, 1949, pages 238 to 243, pages 296-298.

Shaw, Jour. Pharm. and Pharmacol. I, (No. 10) page 700. Reprint 195-100. 

1. A METHOD OF SYNTHESIZING FAT-LIKE SAPONIFIABLE MATERIALS AND ORGANIC ACIDS FROM HYDROCARBONS COMPRISING THE STEPS OF DISPERSING HYDROCARBON IN THE SUBSTANTIAL ABSENCE OF OTHER ORGANIC SUBSTRATE IN AN INORGANIC NUTRIENT MEDIUM FOR MICROORGANISMS, INOCULATING THE RESULTING MIXTURE WITH A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF PENICULLIUM MOLDS, ASPERGILLUS MOLDS, MONILIA YEASTS AND SACCHAROMYCES YEASTS, SUBJECTING THE INOCULATED MIXTURE TO INCUBATING CONDITIONS FOR A SUSTAINED PERIOD OF TIME, COLLECTING THE RESULTING SOLID MATERIAL AND SEPARATING THEREFROM SYNTHESIZED FAT-LIKE SAPONIFIABLE MATERIALS AND ORGANIC ACIDS. 